pET28a-hfYFP-TEV-mScarlet-i
(Plasmid #191196)

• Purpose: Fusion of hfYFP and mScarlet-I separated by a TEV cleavage site for fluorescence-assisted protein purification
• Depositing Lab: Gregory Petsko
• Publication: Campbell et al Nat Methods. 2022 Nov 7. pii: 10.1038/s41592-022-01660-7. doi: 10.1038/s41592-022-01660-7.

Backbone

• Vector backbone: pET28a
• Total vector size (bp): 6769
• Modifications to backbone: Thrombin cleavage site eliminated.
• Vector type: Bacterial Expression

Growth in Bacteria

• Bacterial Resistance(s): Kanamycin, 50 μg/mL
• Growth Temperature: 37°C
• Growth Strain(s): DH5alpha
• Growth instructions: For protein production, transform the plasmid into BL21(DE3) cells and induce expression using IPTG.
• Copy number: High Copy

Gene/Insert

• Gene/Insert name: hfYFP & mScarlet-I fusion
• Species: Synthetic
• Insert Size (bp): 1533
• Promoter: T7 promoter
• Tags / Fusion Proteins:
  • mScarlet-I (C terminal on insert)
  • 6xHis (N terminal on insert)

Cloning Information

• Cloning method: Gibson Cloning
• 5′ sequencing primer: T7 promoter
• 3′ sequencing primer: T7 terminator

Terms and Licenses

• Academic/Nonprofit Terms:
  • UBMTA
• Industry Terms:
  • Not Available to Industry

Depositor Comments

Please refer to the manuscript to understand how to use this expression/purification system.

The streptavidin core domain sequence, constituting amino acids 13-140 of the native protein and responsible for its activity, was synthesized from Thompson & Weber, 1993, without further codon optimization.